Control of kudzu with a fungal pathogen derived from Myrothecium verrucaria

ABSTRACT

Methods for the biological control of kudzu ( Pueraria lobata ) using the fungus  Myrothecium verrucaria  have been developed. In typical applications, conidia of the fungus are applied by means of a liquid surfactant to kudzu in amounts effective to produce plant lesions which kill or suppress the kudzu. A strain of  M. verrucaria  is on deposit with the Department of Biological Sciences, Louisiana Tech University in Ruston, La., and with the patent collection of the International Mycological Institute in surrey, UK, where it has been assigned deposit number IMI 368023.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Kudzu (Pueraria lobata) is a perennial leguminous vine introduced fromJapan that produces a large starchy tuber-like root system. Nowinfesting over 7 million acres of the southeastern United States, andspreading at a rate of 120,000 acres per year, this plant represents aserious threat to productivity in its growing region.

This invention relates to novel methods for the control of kudzu basedupon the application of conidia of Myrothecium verrucaria inherbicidally effective amounts. These methods, due to their systemicnature, do in fact provide superior control of the weed as compared toknown commercial alternatives.

2. Description of the Prior Art

Several methods are known in the art for using microorganisms to controlweeds and other pest plants. As disclosed in U.S. Pat. No. 3,999,973(Daniel et al.), the anthracnose fungus Colletotrichum gloeosporiodeshas been used to control the weed northern jointvetch and another strainof the fungus has been used to control winged waterprimrose.Colletotrichum malvarum has been used to control prickly sida. Thesethree pathogens have been combined to control all three target weeds atonce. In other experimental work, the fungus Alternaria macrospora hasbeen used to control spurred anoda (Anoda cristata) [H. L. Walker, WeedScience, Vol.29, pp.505-507, 1981].

Research activity involving M. verrucaria is noted on page 8 of the IBGNews, Vol.4, No. 1, May 1995 issue. U.S. Pat. No. 4,390,360 (Walker),describes “Control of Sicklepod; Showy Crotaria and Coffee Senna With aFungal Pathogen” using a specific host strain of the fungus Alternariacassiae to produce typical weed lesions which kill or suppress therespective weeds. U.S. Pat. No. 4,419,120 (Walker), discloses “Controlof Prickly Sida, Velvetleaf, and Spurred Anoda With Fungal Pathogens”using a specific host strain of the fungus Fusarium lateritium to killor suppress the respective weeds. U.S. Pat. No. 4,715,881 (Andersen etal.), details “Control of Eastern Black Nightshade With a FungalPathogen” using a strain of Colletotrichum coccodes which is pathogenictowards eastern black nightshade (Solanium prycanthum). U.S. Pat. Nos.4,718,935 and 4,767,441 (Walker et al.), describe a “Method for thePreparation of Mycoherbicide-Containing Pellets” characterized byalginate gel pellets containing living fungus capable of producingconidia when exposed to sufficient light and moisture. U.S. Pat. Nos.4,724,147 and 4,818,530 (Marois et al.), detail the “Preparation ofPellets Containing Fungi for Control of Soilborne Diseases”, in whichfungi are first selected and grown for a time sufficient to produceinoculum. The fungal propagules are harvested, homogenized and dilutedwith sodium alginate solution. Pelletization is then accomplished bydropwise addition of the fungal propagule-alginate mixture into asolution of calcium chloride or calcium gluconate. The resultingalginate gel pellets containing living fungi can then be dried and usedto inoculate agricultural fields infested with soilborne plant diseases.U.S. Pat. No. 5,192,541 (Savage et al.), describes “WeedkillingXanthamonas campestris”, in which novel microorganisms useful incontrolling unwanted grasses and other weeds are discovered through aunique process which involves isolating plant pathogens fromasymptomatic plants. U.S. Pat. No. 5,393,728 (Charudatten et al.),details a “Broad Spectrum Bioherbicide to Control Several Species ofPigweeds”, in which a novel Phomopsis sp. fungus is used as an effectivebroad-spectrum bioherbicide for controlling pigweed.

U.S. Pat. No. 5,747,029 (Walker et al.), hereby incorporated byreference, teaches methods for the biological control of various weedsincluding sicklepod, pigweed, spurred anoda, jimsonweed, and hempsesbania using the fungus M. verrucaria . This work, while showing thatM. verrucaria is effective in controlling a number of varieties of weedsin several different types of important agricultural crops, shows thateffective control of a given species is more the exception than the ruleand that the host range for this fungus was not predictable (Col. 2,lines 66-67). Effective control, where it occurred, also required thepresence of a dew period. The prior art as a whole teaches that fungideveloped as biological herbicides should be restricted in host range toa limited number of plant species.

SUMMARY OF THE INVENTION

We have now discovered means for the effective biological control ofkudzu (Pueraria lobata) using the fungus M. verrucaria. The fungus isisolated, produced, compositioned and applied to kudzu by methodologytaught in U.S. Pat. No. 5,747,029 (Walker et al.), which is hereinincorporated by reference.

In accordance with this discovery, it is an object of the invention toprovide means for the effective biological control of kudzu.

Other objects and advantages of the invention will become readilyapparent from the ensuing description.

DEPOSIT OF BIOLOGICAL MATERIAL

M. verrucaria(Alb. and Schwein) Ditmar ex Fr. is on deposit with theDepartment of Biological Sciences, Louisiana Tech University in Ruston,La., at the United States Department of Agriculture, AgriculturalResearch Service, Southern Weed Science Research Unit in Stoneville,Miss.; and was placed on deposit with the International MycologicalInstitute, Bakeham Lane, Egham Surrey, UK, on May 18, 1994, as IMInumber 361690. The deposit was placed in the patent collection of theInternational Mycological Institute on Jun. 21, 1995, under the terms ofthe Budapest Treaty, and has been assigned accession number IMI 368023.

According to M. Tulloch, (The Genus Myrothecium Tode ex Fr. MycologiicalPapers 130, 1-42, 1972), M. verrucaria is described as follows: “Sporemass wet, black, convex surrounded by wet floccose margin. Sporesbroadly fusiform, one end pointed the other protruding and truncate, inerythrosin and NH₄ solution with a fantailed appendage on the pointedend, 6.5-8×2.3-5 microns.”

The M. verrucaria used in this invention was isolated from diseasedplants of sicklepod, a new host record for this fungus.

DETAILED DESCRIPTION OF THE INVENTION

The fungus may be cultured by conventional means, such as thosedisclosed in U.S. Pat. No. 5,747,029 (Walker), hereby incorporated byreference. Conidia produced are then formulated in aqueous solution andcompositioned with a surfactant such as Silwet L-77 (trademark); asilicone-polyether copolymer spray adjuvant, OSI Specialties, Inc.,Charlotte, N.C. Other useable surfactants include Tween-20 [oxysorbic(20 POE) (polyoxyethylene sorbitan monooleate)], Tween-80 (Polysorbate80) and Sterox [nonoxynol (9 to 10 POE)[a-(p-onylphenyl)-w-hydroxypoly(oxyethylene)]]. Useable compositionalconcentrations of the conidia range from about 2×10⁶ to about 2×10⁸conidia/ml of solution, with the surfactant being present in amountsranging from about 0.02% to about 0.4% (vol/vol), preferably about 0.1to about 0.3% (vol/vol). Application is made to the kudzu at a rateranging from about 5 to about 100 gallons per acre, preferably about 40to about 60 gallons per acre. Effective control of the kudzu is hereindefined as achieving a biocidal rate of 90% or greater within 14 days ofapplication. Efficacy does not require the presence of a dew period asindicated by growth chamber and greenhouse tests, and is similarlytolerant under field conditions to temperatures up to about 40° C.

The experimental parameters used in examples cited for this inventionare not intended to limit the scope of this invention. Modification offactors such as inoculum concentrations, parameters for inoculumproduction, surfactants, application methods, and other factors, wouldbe expected to influence efficacy of this invention. Parameters wereselected to enable detection of interactions, to document therelationship of this invention to the prior art, and to illustrate thatthe unique and surprising characteristics of this invention were notobvious and could not have been predicted from the prior art.

EXAMPLE 1

Inoculum Production

Inocula (conidia) of M. verrucaria for all experiments were produced inpetri dishes containing Difco potato dextrose agar (PDA). Agar surfaceswere flooded with 1 ml of a M. verrucaria conidia suspension containing2×10⁶ conidia/ml. The dishes were inverted on open-mesh wire shelves andincubated at 25° C. for 5 days in fluorescently lighted incubators. Theresulting conidia were rinsed from the cultures with sterile, distilledwater, and were adjusted to the desired concentrations by addingdistilled water. Conidia counts and concentrations were estimated withhemacytometers. PDA inoculated with conidial suspensions produced fungallawns after 5 days. When conidia were harvested by flooding the cultureswith 10 ml of distilled water, each culture produced approximately 8×10⁸conidia.

Test Plant Propagation

Kudzu seedlings were grown from seed in 10 cm plastic pots containing a1:1 (w/w) commercial potting mix/soil combination supplemented with acontrolled release 13:13:13 (N:P:K) fertilizer. Temperatures in thegreenhouse ranged from 28° C. to 32° C. with 40 to 60% relativehumidity. The photoperiod was approximately 14 hours, with 1600 to 1800μmol/m²/s photosynthetically active radiation (PAR) at midday, asmeasured with a light meter.

Effect of Inoculum Concentration and Plant Growth

Seedlings in either the cotyledonary, first-to-third leaf,fourth-to-sixth leaf, or seventh-to-eighth leaf growth stage wereinoculated by aerosol sprayers until foliage was fully wetted witheither 0, 2×10⁵, 2×10⁶, 2×10⁷, or 2×10⁸ conidia/ml contained in 0.2%Silwet 1-77 surfactant. Following inoculation, the plants were incubatedon greenhouse benches and monitored for disease development. Greenhouselighting and temperature conditions were as described previously.Experimental units consisted of groups of 10 plants. The treatments werereplicated three times and the experiment was conducted twice. Arandomized complete block experimental design was utilized, and the datawere analyzed using regression analysis and 95% level confidence limits.

As shown in Table I, mortality was significantly increased at all growthstages by increasing the inoculum concentration. Greater overallmortality was achieved with inoculum concentrations of 2×10⁷ and 2×10⁸conidia/ml than with lower concentrations tested. An inoculumconcentration of 2×10⁶ conidia/ml killed 40-50% of the weeds in thecotyledonary growth stage, but was less efficacious on larger plants.Seedlings in the cotyledonary, first to third leaf, and fourth to sixthleaf stages were effectively killed with 1×10⁷ conidia/ml, but largerplants required an order of magnitude larger inoculum concentration tobe 90% controlled.

TABLE I Effect of Plant Growth Stage and Inoculum Concentration onControl of Kudzu by Myrothecium verrucaria Kudzu Mortality (%)¹ InoculumConcentration (Spores/ml)² Number of Leaves 0 2 × 10⁴ 2 × 10⁵ 2 × 10⁶ 2× 10⁷ 2 × 10⁸ Cotyledon 0 10(4) 12(3) 48(6) 100 100 1-3 0 5(3) 6(0)35(5) 94(4) 100 4-6 0 0 0 35(4) 85(6) 95(4) 7-8 0 0 0 21(4) 60(5) 84(5)¹Values represent an average obtained from two experiments with groupsof 10 plants in each experiment. Values in parentheses are standard meanerrors. ²Kudzu plants were sprayed until runoff occurred.

Effect of Incubation Temperature

Kudzu plants in the cotyledonary to first true leaf stages of growthwere inoculated by aerosol sprayers until the foliage was fully wettedwith suspensions containing 2×10⁷ conidia/ml plus 0.2% Silwet L-77surfactant. Control plants were sprayed with 0.2% surfactant only.Immediately following inoculation, the plants were placed in Shearer(Rheem Mfg. Co., Weaverville, N.C.) growth chambers at constantday/night temperatures of 10° C., 15° C., 20° C., 25° C., 30° C., 35° C.or 40° C. Photoperiods were 14 hour day/10 hour night with approximately900 μmol/m²/s PAR. Disease development was monitored daily. After 14days following inoculation all 10 plants of each experimental unit, bothliving and dead, were excised at the soil line, combined, and dried (80°C. for 7 days) for dry weight determinations. A randomized completeblock experimental design was utilized, and the data were analyzed using95% confidence limits.

As shown in Table II, pathogenesis and mortality occurred at alltemperatures that were tested. Higher temperatures promoted greaterdisease development and weed control. Disease symptomatology wascharacterized by necrotic flecking which occurred within 6 hoursfollowing treatment at incubation temperatures of 30-40° C. with slowerdisease development at lower temperatures. Disease symptoms progressedfrom inoculated cotyledons and leaves to produce stem lesions within 48hours. This indicates that the invention could be used even in midsummerwhen similar temperatures in kudzu-infested regions of the southeasternUnited States occur.

TABLE II Effect of Incubation Temperatures on Mortality and Dry WeightReduction of Kudzu by Myrothecium verrucaria ¹ Incubation Temp. KudzuMortality Dry Weight (° C.) (%)² Reduction (%) 10 8(4) 10(3) 15 18(2)20(4) 20 38(5) 42(4) 25 72(6) 80(5) 30 92(4) 95(3) 35 100(0) 100(0) 40100(0) 100(0) ¹Plants in the cotyledonary stage of growth sprayed untilrunoff occurred at a concentration of 2.0 × 10⁷ spores/ml. ²Valuesrepresent an average obtained from two experiments with groups of 10plants in each experiment. Values in parentheses represent mean standarderrors at p = 0.05.

Field Experiments

Kudzu seedlings in the cotyledonary to first leaf growth stage weretransplanted into 0.5 m² field microplots in separate experiments. Eachplot consisted of 10 seedlings. The plants were allowed to acclimate tofield conditions for one week prior to treatment. Treatments consistedof 2×10⁷ conidia/ml in distilled water, 2×10⁷ conidia/ml in 0.2% SilwetL-77, distilled water only, and 0.2% Silwet L-77 only. The plants weresprayed until fully wetted (approximately 3 ml/plant). Applications weremade at midday with a handheld pressurized sprayer. The plants weremonitored for disease development at 5 day intervals for 15 days, thenharvested for dry weight determinations as described previously for thegrowth chamber experiments. A randomized complete block design wasutilized, and the treatments were replicated three times. Data from thetwo experiments were pooled following subjection to Bartlett's test forhomogeneity (Steele and Torrey, 1980) and were analyzed using theanalysis of variance. Treatment means were separated using Duncan'smultiple range test.

In the microplot experiments, kudzu plants treated with thefungus/surfactant mixtures exhibited leaf and stem necrosis within 24hours following inoculation, with mortality occurring within 96 hours.After 7 days, 100% of the inoculated plants had been killed in plotstreated with M. verrucaria/Silwet L-77 mixtures. The fungus sporulatedprofusely on infected tissue and was easily reisolated. No visibledamage was observed on plants in plots treated with the fungus indistilled water only, 0.2% Silwet L-77 only, or untreated controls, andno dry weight reductions occurred in any of these treatments.

EXAMPLE 2

A field test was established in a site that was heavily infested with anaturally occurring kudzu population. The plants were vigorous and hadnot yet flowered. Treatments consisted of: 1) 2×10⁶ conidia/ml indistilled water; 2) 2×10⁷ conidia/ml in distilled water; 3) 2×10⁶conidia/ml in 0.2% Silwet L-77 surfactant; 4) 2×10⁷ conidia/ml in0.2%,Silwet L-77 surfactant; 5) 0.2% Silwet L-77 surfactant only, and 6)untreated control. Spray volumes were applied at 450 L/hectare withbackpack sprayers. Visual ratings based the percentage of necrotic kudzutisses in treated plots as compared to untreated control plots were usedto assess weed control at weekly intervals for 4 weeks. The test wasarranged in a completely randomized design with 3 replications.

Kudzu was controlled 100% after 14 days in plots treated withfungus/surfactant mixtures applied at 2×10⁷ conidia/ml, with no visualsymptoms or weed control occurring in any other treatment. After 4weeks, vines from untreated plot margins had begun to spread intotreated areas where kudzu had been defoliated, but no new leaf growthoccurred on vines that had been considered “killed”.

While the preferred embodiments have been described above, it will berecognized and understood that various modifications may be made in theinvention and the appended claims are intended to cover all suchmodifications which may fall within the spirit and scope of theinvention.

We claim:
 1. A method for the biological control of kudzu comprising theapplication of a herbicidally effective amount of Myrothecium verrucariathereto.
 2. The method of claim 1 wherein said Myrothecium verrucaria isapplied in the form of an aqueous composition containing a liquidsurfactant.
 3. The method of claim 2 wherein said surfactant is selectedfrom the group consisting of a silicone-polyether copolymer sprayadjuvant, oxysorbic (20 POE) polyoxyethylene sorbitan monooleate,Polysorbate 80 and nonoxynol (9 to 10 POE).
 4. The method of claim 1wherein said Myrothecium verrucaria has the identifying characteristicsof strain IMI
 368023. 5. The method of claim 1 wherein said Myrotheciumverrucaria is applied in the form of conidia.